Process for producing 5-fluorouracil riboside and 6-mercaptopurine riboside

ABSTRACT

A process for producing 5-fluorouracil or 6-mercaptopurine riboside by fermentation which comprises culturing a microorganism belonging to Bacillus subtilis or Streptomyces fradiae in an aqueous nutrient medium under aerobic conditions. In the case of 5-fluorouracil riboside, 5-fluorouracil is added to the medium and in the case of 6-mercaptopurine riboside, 6mercaptopurine is added to the medium. The products have been reported as being useful as antagonistic agents of nucleic acid metabolism and as anti-cancer agents.

United States Patent Nakayama et al.

[451 Mar. 21, 1972 PROCESS FOR PRODUCING 5- FLUOROURACIL RIBOSIDE AND 6-MERCAPTOPURINE RIBOSIDE Inventors: Kiyoshi Nakayama, Sagamihara-shi;

Takashi Nara, Tokyo; Masanaru Misawa, Kawasaki-shi; Toshio Komuro,Machidashi, all of Japan Assignee: Kyowa Hakko Kogyo Co., Ltd., Tokyo,

Japan Filed: July 15, 1968 Appl. No.: 744,690

Related U.S. Application Data Continuation of Ser. No. 565,089, July 14,1966, abandoned.

Foreign Application Priority Data July 19, 1965 Japan ..40/43227 U.S.Cl. ..l95/28 N Int. Cl. Cl2d 13/06 Field of Search l 95/28 N PrimaryExaminer-Alvin E. Tanenholtz Attorney-Craig, Antonelli & Hill [57]ABSTRACT A process for producing 5-fluorouracil or 6-mercaptopurineriboside by fermentation which comprises culturing a micro organismbelonging to Bacillus subtilis or Streptomycesfradiae in an aqueousnutrient medium under aerobic conditions. In the case of S-fluorouracilriboside, 5-fluorouracil is added to the medium and in the case of6-mercaptopurine riboside, 6- mercaptopurine is added to the medium. Theproducts have been reported as being useful as antagonistic agents ofnucleic acid metabolism and as anti-cancer agents.

9 Claims, N0 Drawings PROCESS FOR PRODUCING S-FLUOROURACIL RIBOSIDE ANDG-MERCAPTOPURINE RIBOSIDE CROSS-REFERENCE TO RELATED APPLICATIONfiuorouraeil riboside ti-mereaptopurine riboside M These compounds arethe ribosides of 5-fluorouracil and '6- mercaptopurine. The lattercompounds have received attention as anti-cancer agents (for example, U.S. Pat. Nos.

2,721,866 and 2,724,711 of Hitchings et al.). The compounds of thepresent invention are of importance as antagonistic agents of nucleicacid metabolism and as anti-cancer agents (for example, A. Lindher etal., Experimental Cell Research, Supplement Vol. 9, p. 485 511, 1963 forS-fluorouracil riboside, and Brit. Patent 936,654 of Wellcome FoundationLtd., 1. Goodmanet al., Federation Proceedings Vol. 14, p. 219, 1955 for-mercaptopurine'riboside).

One of the objects of the present invention is to provide a process forthe productionof 5-fluorouracil riboside and 6- mercaptopurine ribosideby fermentation which may be carried out in an efficacious and simplemanner.

Another object of the present invention is to provide a process forproducing S-fluorouracil riboside and 6-mercaptopurine riboside byfermentation which gives thesproduct in high purity and good yield.

A further object of the invention is to provide a process for producing5-fluorouracil riboside and "fi-mercaptopurine riboside by fermentationwhich may be carried out advantageously on an industrial scale at lowcost to give a high yield of product.

These and other objects and advantages of the-presentinvention willbecome apparent to those skilled in the artfrom a consideration of thefollowing specification and claims.

In accordance with the present invention, it hasbeen found thatremarkably large quantities of S-fluorouracil riboside or-mercaptopurine riboside are accumulated in the fermentation liquor andmay be recovered therefrom if fermentation or culturing is carried outwith micro-organisms capable of producing these compounds, such asBacillus-subtilis, Streptom yces fradiae and the like,and addingS-fluorouracil or 6-mer captopurine, depending upon which product isdesired, to the culture medium. This latter addition may be made at anytime during the culturing. The nucleoside corresponding to therespective base added to the medium thus accumulates in large quantitiesas a result thereof.

The general conditions of culturing are those conventionally used in theart for fermentation processes. Either a synthetic culture medium or anatural nutrient medium is suitable in the present invention as long asit contains the essential nutrients for the growth of the strainemployed. Such nutrients are well known in the art and includesubstances such as a carbon source, a nitrogen source, inorganiccompounds and the like which are utilized by the micro-organism employedin appropriate amounts. Thus, as a carbon source, carbohydrates such asglucose, fructose, maltose, sucrose, starch, starch hydrolysate, etc.,may be employed. Of course, other carbon sources such as glycerol,organic acids, glutamic acid and the like may be employed. A singlecarbon source may be used, or a mixture of two or more. As a nitrogensource, various kinds of inorganic or organic salts or compounds, suchas urea or ammonium salts such as ammonium chloride, ammonium sulfate,ammonium nitrate, ammonium phosphate, etc., or natural substancescontaining nitrogen, such as cornsteep liquor, yeast extract, meatextract, peptone, fish meal, casein hydrolysate, casamino acid, fishsolubles, rice bran extract, etc., may be used. Again, thenitrogen-containing substances may be used either singly or incombinations of two or more. lnorganiccompounds which may be added tothe culture medium include salts such as magnesium sulfate, sodiumphosphate, potassium dihydrogen phosphate, potassium monohydrogenphosphate, iron sulfate or other iron salts, manganese chloride, calciumchloride, as well as other appropriate salts of manganese, zinc and thelike.

Fermentation is carried out under aerobic conditions, such as aerobicshaking of the culture or with stirring of a submerged culturewithaeration, at a temperature of about 20 to 40C. and a pH of about 5.0to 9.0. After about 2 to 10 days of culturing, remarkably large amountsof the particular nucleoside desired, whether it be 5-fluorouracilriboside or 6- mercaptopurine riboside depending upon the particularbase added to the culture medium, are accumulated in the culture liquoras well as in the cell bodies themselves.

After the completion of culturing, the 5-fluorouracil riboside or6-mercaptopurine riboside may be recovered from the fermentation liquorby conventional means, such as ion exchange resin treatment, extractionwith solvents, precipitation with metallic salts, adsorption, and thelike.

The amount of S-fluorouracil or 6-mercaptopurine base to :be addedranges from about 0.1 to 10 mg./ml. of culture medi- The followingexamples are given merely as illustrative of the present invention andare not to be considered as limiting. Unless otherwise noted, thepercentages therein are by weight per liter of water.

EXAMPLE] Bacillus subtilis ATCC 19062 is used as the seed bacterium.This strain is cultured in a culture medium consisting of 2% of glucose,1% of peptone, l% of yeast extract, 0.25% of NaCl and 30 Lg/l. ofbiotinat 30C. for 24 hours. The pH of the culture medium is adjusted to 7.3.

The resultant seed medium is inoculated in an amount of 10 percent byvolume into the following fermentation medium composition:

[0% glucose ll yeast extract 1% NH, ,so, 0.6% K,HPO, 0.6% KH,P0, 0.6% MsonHp 2% CaCO resultant S-fluorouracil riboside is recovered bychromatography on Dowex I (trade name of a polystyrene strongly anionicion exchange resin of the Dow Chemical Co.) (formate type).

EXAMPLE II The same seed strain, seed medium and fermentation medium asdescribed in Example I are employed. After 68 hours of culturing,fi-mercaptopurine is added to the medium, instead of S-fluorouracil, togive a concentration thereof of 2.0 mg./ml. After 120 hours ofculturing, 89 mg./ml. of 6-mercaptopurine riboside is accumulated in thefermentation liquor. The resultant 6-mercaptopurine riboside isrecovered from the fermentation liquor by a combination of achromatography procedure on Dowex I (trade name of the polystyrenestrongly anionic exchange resin of the Dow Chemical Co.) (formate type)and a carbon adsorptiondesorption method.

EXAMPLE lll Srreplomyces fradiae ATCC 19063 is employed as the seedbacterium. The same culture medium and fermentation medi um as describedin Example 1 are used. After 68 hours of culturing, 6-mercaptopurine isadded to the culture liquor in an amount so as to give a concentrationthereof of 2.0 mg./ml. therein. After 120 hours of culturing, 0.42mg./ml. of 6-mercaptopurine riboside is found to be accumulated in thefermentation liquor.

The invention being thus described, it will be obvious that the same maybe varied in many ways. Such variations are not to be regarded as adeparture from the spirit and scope of the invention, and all suchmodifications as would be obvious to one skilled in the art are intendedto be included herein.

What is claimed is:

1. A process for producing a compound selected from the group consistingof 5-fluorouracil riboside and fi-mercaptopurine riboside which consistsessentially of culturing a micro-organism capable of producing saidcompound and belonging to Bacillus subtilis or Streptomycesfradiae in anaqueous nutrient medium under aerobic conditions in the presence of abase selected from the group consisting of 5-fluorouracil, in the caseof the production of 5-fluorouracil riboside, and 6-mercaptopurine, inthe case of the production of -mercaptopurine riboside, and recoveringthe resultant compound from the fermentation liquor.

2. The process of claim 1, wherein said base is added to the mediumprior to the initiation of culturing.

3. The process of claim 1, wherein said base is added to the mediumduring culturing.

4. The process of claim 1, wherein said culturing is carried out at atemperature of from about 20 to 40 C. and a pH of about 5.0 to 9.0.

5. The process of claim 4, wherein said micro-organism is Bacillussublilis ATCC 19062.

6. The process of claim 4, wherein said micro-organism isStreptomycesfradiae ATCC 19063.

7. The process of claim 4, wherein said compound is 5- fluorouracilriboside and said base is S-fluorouracil.

8. The process of claim 4, wherein said compound is 6-mercaptopurineriboside and said base is 6-mercapt0purine.

9. The process of claim 4, wherein the amount of said base ranges fromabout 0.1 to 10 mg./ml. ofculture medium.

2. The process of claim 1, wherein said base is added to the mediumprior to the initiation of culturing.
 3. The process of claim 1, whereinsaid base is added to the medium during culturing.
 4. The process ofclaim 1, wherein said culturing is carried out at a temperature of fromabout 20* to 40* C. and a pH of about 5.0 to 9.0.
 5. The process ofclaim 4, wherein said micro-organism is Bacillus subtilis ATCC
 19062. 6.The process of claim 4, wherein said micro-organism is Streptomycesfradiae ATCC
 19063. 7. The process of claim 4, wherein said compound is5-fluorouracil riboside and said base is 5-fluorouracil.
 8. The processof claim 4, wherein said compound is 6-mercaptopurine riboside and saidbase is 6-mercaptopurine.
 9. The pRocess of claim 4, wherein the amountof said base ranges from about 0.1 to 10 mg./ml. of culture medium.